Limited splicing of precursor RNA is an essential component of the retrovirus life cycle. Splicing of viral primary transcripts must be controlled since this RNA is required for expression of structural proteins and as genome for progeny virions. A substantial amount of unspliced retrovirus RNA accumulates in an infected cells. Two elements distinct from the splice sites play a role in maintaining high levels of unspliced RNA in Rous sarcoma virus: i) An element in the gag gene termed the negative regulator of splicing (NRS) controls splicing from the common 5' splice site to both the env and src 3' splice sites and ii) A second element near the src 3' splice site appears to control src splicing specifically. Since viral proteins are not required, host factors must mediate these events. The goal of the work is to identify and characterize cellular proteins important in this novel form of gene expression regulation employing in vivo and in vitro strategies. Proteins that specifically interact with the NRS will be identified using UV crosslinking, mobility shift assays, and chromatographic approaches. Roles for NRS-binding proteins (NBPs) in splicing inhibition will be assessed in vitro and in vivo using mutant elements defective for inhibition. The NRS- binding proteins will be purified, sequenced, and cDNAs encoding them identified to allow subsequent biochemical experiments to address their function and subcellular distribution. Similar in vivo and in vitro experiments will be performed with the src element to elucidate its mode of action. Since its function may depend on structure, the secondary structure of the src inhibitory element will be determined chemically and enzymatically. Proteins important for src splicing inhibition will be sought and compared to those necessary for NRS activity to reveal potential mechanistic parallels between the two elements. Thr NRS has also been shown to influence polyadenylation efficiency in the 3'LTR; NRS mutations facilitate read through into host DNA, which can result in oncogene activation. In vitro reactions will be used to further study NRS poly(A) promotion. The possibility that the NRS recruits polyadenylation factors will be investigated using assays designed to detect interactions between NBPs and components of the polyadenylation machinery. Understanding retroviral splicing control by interactions of host-cell factors with viral cis-acting elements may yield insights into regulation of cellular mRNA processing as well.